Inactivating mutations of PHEX, which underlie XLH, modulate expression of the disease, in part, by regulating serum FGF-23 levels. Our recent studies have determined that: 1) the physiologically relevant site for expression of the Phex mutation is the osteoblast; and 2) the bone mineralization abnormality in Hyp-mice and various targeted Phex knockout mice ranges from mild P-dependent disease to severe P refractory disease, the latter of which is associated with increased osteoblast Sost mRNA production. Despite these discoveries, the mechanism whereby inactivation of Phex alters Fgf-23 degradation and/or production and consequently serum hormone levels remains unknown, as does the mechanism by which such mutations enhance Sost mRNA production. However, our recent advances have provided the framework to understand these regulatory processes. These advances indicate that loss of function Phex mutations in Hyp-mice limit production of the chaperone protein, 7B2, in bone/osteoblasts, reducing 7B2?SPC2 activity. Suppressing this enzyme function results directly in impaired Fgf-23 degradation and, through down-stream effects, increased Fgf-23 production, both of which contribute to increased circulating Fgf-23 levels. In additional studies, we discovered that the limited availability of 7B2 also enhances bone/osteoblast production of Sost mRNA .and Sclerostin, an increased concentration of which impairs bone mineralization. In the current application, we propose to determine if: 1) the down-stream PHEX/Phex dependent alterations of 7B2 and 7B2?SPC2 regulate expression of the HYP phenotype through modulation of Fgf-23 degradation and production and Sclerostin production; and 2) 7B2 and 7B2?SPC2 are therapeutic targets for successful treatment of XLH. We will employ a series of experimental strategies to increase bone 7B2 protein and/or 7B2?SPC2 enzyme activity to confirm the central role of the chaperone protein in genesis of the Fgf-23 dependent renal abnormalities, characteristic of the HYP phenotype and to assess if normalization of 7B2 and/or 7B2?SPC2 activity is a successful treatment strategy for XLH. In complementary studies, using the same experimental strategies we will investigate the potential role of Sclerostin in the bone mineralization defect in Hyp-mice. To confirm such Sclerostin effects on bone mineralization, we will also explore the effects of antibody neutralization of Sclerostin on bone mineralization in appropriate hypophosphatemic animal models. In related studies during the last funded cycle, we used animals with transgenic over-expression of Npt28 to document a role for P transport in regulation of renal 25(OH)D-1a-hydroxylase activity in Hyp-mice. As an extension of these investigations in the current application, we propose experiments to determine if renal P transport, an apparent modulator of renal 25(OH)D-1a-hydroxylase activity in Hyp-mice, universally regulates 1,25(OH)2D production. We will use conditional knockout of Npt2 to test the effect of renal P transport on enzyme function in normal animals. In complementary studies we will investigate if the impaired renal P transport induced post- transcriptional defect in vitamin D metabolism is governed by abnormal phosphorylation dependent redirection of 25(OH)D-1a-hydroxylase from the ER to the mitochondria Our studies are significant, as they will clarify dependence of the HYP renal phenotype on FGF-23 effects, while enhancing our understanding of vitamin D metabolism and P homeostasis and bringing new perspective to the bone mineralization abnormality in XLH. These advances may lead to creating a new effective treatment strategy for this disease. PUBLIC HEALTH RELEVANCE: A poor understanding of the pathophysiology of the Vitamin D Resistant Diseases in man has long precluded optimal care of these disorders. The research proposed in this study will significantly increase our understanding of the pathophysiological cascade of events underlying the archetypal Vitamin D Resistant Disease, X-Linked Hypophosphatemia (XLH). Such advances will lead to new therapeutic approaches not only to XLH but to related diseases and promise to improve the outcomes of therapy.